(54g) Determination of Size, Size Distribution and Refractive Index of Artificial and Biological Microparticles

For the approval of
clinical studies, it is mandatory to validate the production process of
artificial oxygen carriers by reliably specifying the properties of the microparticles
used like the newly developed blood substitute HbMP-700 (1-3). In particular, besides shape, size and size distribution, the
amount of hemoglobin (Hb) and methemoglobin (metHb) content is essential.

We present a method based
on the simultaneous measurement of collimated and diffuse transmission spectra in
the visible and ultraviolet range and numerical analysis to infer the
wavelength-dependent refractive index, size and size distribution of microparticles.

For the validation of
the method, we used aqueous suspensions of monodisperse polystyrene
microspheres of known concentration, determined by flow cytometric
measurements. The related data analysis is carried out for synthetic and
experimental data. For homogeneous spheres, Mie theory is applied to compute the
solution for the scattering of a plane electromagnetic wave by a particle and
to calculate the extinction cross section C_ext, i.e. the shadow cast by the particle as a function
of the vacuum wavelength λ, the particle size, and the complex refractive index n(λ). For dilute suspensions of particles, the measured
quantity is an ensemble-averaged extinction cross section ⟨C_ext⟩. This
average is obtained by integrating over the particles' size distribution, which
may not be known a priori. From the resulting spectral extinction
cross sections, the complex refractive index and the mean and standard
deviation of the size distribution can be inferred by means of nonlinear
least-squares optimization. In order to reduce the
number of free parameters of the problem to a feasible amount, n(λ) is
represented in a set of harmonic-oscillator-like resonance functions. The
refractive index of polystyrene microspheres obtained is in good agreement with
literature values for the bulk material, the mean and standard deviation of the
distribution of particle size D are determined with an uncertainty in the range of a
few nanometers. For example, for microspheres of mean diameter mean(D) = 2µm and distribution width std(D) = 9.4nm the uncertainty of the mean is u{mean(D)} = 1.2nm and the uncertainty of the distribution width
amounts to u{std(D)} = 0.4nm.

In a second step, we
applied the method to characterize HbMP-700 microparticles, approximately featuring
a prolate spheroidal shape with 700nm length and
400nm width. Besides shape, size and size distribution, the amount of hemoglobin
(Hb) and methemoglobin (metHb)
content is essential. The relative concentrations of oxyHb,
deoxyHb, metHb are
accessible from spectral extinction and diffuse transmission measurements,
since the different Hb variants exhibit different
wavelength-dependent refractive indices. The method to infer size and
refractive index described above can be applied to these non-spherical particles, since
the deviation of their light scattering properties from those of spheres turns
out to be small. The results based on the optical method will be compared to
alternative procedures for HbMP particle
characterization with respect to their size and size distribution, i.e.
electron microscopy, centrifugation and dynamic light scattering. In addition,
x-ray spectrometry will be used to detect the ratio between Fe²⁺
(oxy/dexyHb) and Fe³⁺ (metHb)
as well as the iron concentration.

References

1)    
Novel hemoglobin
particles--promising new-generation hemoglobin-based oxygen carriers. Bäumler H, Xiong Y, Liu
ZZ, Patzak A, Georgieva R. Artif Organs. 2014
Aug;38(8):708-14. doi: 10.1111/aor.12331.

2)    
Nonvasoconstrictive
hemoglobin particles as oxygen carriers. Xiong Y, Liu
ZZ, Georgieva R, Smuda K, Steffen A, Sendeski M, Voigt A, Patzak A, Bäumler H. ACS Nano. 2013 Sep
24;7(9):7454-61. doi: 10.1021/nn402073n.

3)    
Hemoglobin-based oxygen
carrier microparticles: synthesis, properties, and in vitro and in vivo
investigations. Xiong Y, Steffen A, Andreas K,
Müller S, Sternberg N, Georgieva R, Bäumler H. Biomacromolecules. 2012 Oct 8;13(10):3292-300. doi: 10.1021/bm301085x.